以血凝素为靶点的H1N1流感病毒融合抑制剂的高通量筛选的方法学研究

H1N1 influenza virus is one of the major threat to the human health in current, but the prevention and treatment situation of H1N1 influenza is not optimistic, and the only anti-H1N1 influenza drugs tamiflu also faces a rapid increase in resistance. Therefore, it is urgent to develop other drugs with different role. Hemagglutinin (HA) plays crucial roles in the early stage of influenza virus, including virus binding to host receptors and membrane fusion. HA is a trimeric glycoprotein in nature and can be cleaved into HA1 and HA2 subunit. After binding to receptor through HA1, virus is taken into cells by endocytosis. Within the endosomal compartment, the virion is exposed to the increasing acid pH 5-6, and the HA2 subunit undergoes an irreversible conformation change from involving extrusion of the fusion peptide (FP) from the interior of the HA2 at the neutral-pH structure toward the endosomal membrane,promoting fusion of the viral and endosomal membranes. According to this, HA is of great potential to be an antiviral drug target, especially the more conserved HA2 subunit.This project is research into the stage in the role of virus (targeted influenza virus coated protein) of a flow of drugs, the influenza virus infection target cells by the process of coated protein hemagglutinin (HA) mediated, its cross membrane and the HA2 inside the cell in the body acidic conditions occur conformation change, mediated virus inside the cell membrane and the integration of body mask. Therefore, according to our research experience in virus enter inhibitors of HIV, SARS virus, we want to establish the high-throughput screening methods for screening the compounds inhibited formation of the six-helix bundle and the N-terminal cap structure in the HA2 domain of AIV, and to develop a cell-based pharmacological model by using the H1N1 pseudotyped viral system, a MDCK cell infected model for in vitro infection by live H1N1 virus, and a mouse model for in vivo infection by live H1N1 virus, and to screen compounds which can specifically inhibit the virus entry.

甲型H1N1流感病毒（甲流）是当前人类健康面临的重大威胁之一，但甲流的预防和治疗形势很不乐观，仅有的抗甲流药物达菲也面临着耐药性迅速增加的问题，因此，急需研制作用于其它靶点的抗甲流药物。在病毒入侵过程中，其包膜糖蛋白血凝素（HA）的HA2亚基介导病毒膜与胞内体膜的融合，即在胞内体的低pH环境下，HA2发生构象改变，插到胞内体膜上，同时三分子HA2形成类似HIV gp41的六螺旋束的结构，从而使病毒膜与胞内体膜发生融合。因此流感病毒与靶细胞结合融合过程可作为药物的作用靶点，开发病毒进入抑制剂。为此，本课题根据我们在禽流感病毒、HIV、SARS病毒进入抑制剂研究领域积累的经验，建立抑制甲流病毒HA2亚基在酸性环境下发生构象变化，尤其是抑制六螺旋束结构形成的高通量筛选方法，并筛选小分子化合物库，寻找能有效抑制甲型H1N1流感病毒进入靶细胞的活性小分子，为开发病毒进入抑制剂奠定基础。

项目背景：甲型H1N1流感病毒（甲流）是流感病毒引起的急性呼吸道传染病，血凝素(Hemagglutinin, HA)是流感病毒的膜外蛋白，由HA1和HA2两个亚基组成，在融合过程中三分子HA2会形成类似六螺旋束的结构。根据血凝素的晶体结构，我们推测小分子化合物若能阻止流感病毒HA2“发卡”样融合结构的形成，尤其是抑制六螺旋束结构形成，有可能具有抗流感病毒感染的作用。为此，本研究采用我们在HIV和SARS-CoV研究领域长期积累的经验，建立抑制六螺旋束结构形成的高通量筛选方法，大规模筛选小分子化合物库，寻找病毒进入抑制剂先导化合物，为开发抗H1N1流感药物奠定基础。.主要研究内容:.1）建立抑制甲型H1N1流感病毒HA2亚基在酸性环境下发生构象变化，尤其是抑制六螺旋束结构形成的高通量筛选方法，并筛选小分子化合物。.2）大规模筛选小分子化合物库，寻找具有特异性抑制甲型H1N1流感病毒进入靶细胞的病毒进入抑制剂先导化合物。.3）利用H1N1型活病毒感染MDCK模型和H1N1型活病毒感染小鼠模型进行验证，寻找能有效抑制H1N1型流感病毒感染的活性分子，研究其抗流感的作用。.重要结果:.1）成功建立了H1N1型流感假病毒感染模型，寻找到两个能有效抑制H1N1型流感的进入抑制剂---吡啶类似物ARC-36和ARC-27。.2）MDCK细胞毒性实验显示ARC-36的CC50为525.4 µM，滴鼻感染前滴鼻给药组和感染后腹腔注射8天组小鼠的平均存活时间分别为8.8天和8.6天，与感染前正常对照组比较差异有统计学意义(p<0.01)。.3)药物的药代动力学研究及毒性研究：ARC-36的保留时间为6.3 min，血浆C-t曲线呈二室模型，消除半衰期(t1/2)β为31.6 min。.4）进一步确认ARC-36的抗H1N1作用：测定了 14个突变位点假病毒感染MDCK细胞的荧光发光值，发现HA2的N502、E1052、T1072、F1102对病毒的进入起着关键的作用。.关键数据及其科学意义:.1）假病毒体系HA-NA/HIV筛选出的化合物ARC-36抑制H5N1禽流感假病毒活性的IC50值为4.00±0.38 µM，且作用在且作用在病毒进入阶段。.2）分子动力学模拟显示ARC-36与R1062和T1072发生氢键相互作用。.3以ARC-36为先导化合物进行结构修饰，合成了一系列衍生物。